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Berberine (BBR), an isoquinoline alkaloid isolated from Rhizoma coptidis, is reported to possess antiviral activity. Our previous study has shown that BBR alleviates Coxsackievirus B3 (CVB3) replication in HeLa cells. However, the anti-CVB3 activity of BBR is still unclear in vivo. In this study, we explored the effect of BBR on CVB3-induced viral myocarditis in mice. These results demonstrated a beneficial effect of BBR on alleviating CVB3-induced myocarditis in vivo, which sheds new light on the utility of BBR as a therapeutic strategy against CVB3-induced viral myocarditis. This article is protected by copyright. All rights reserved.

O(2)- and O(4)-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O(2)- and O(4)-n-butylthymidine (O(2)- and O(4)-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O(2)-nBudT- and O(4)-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O(2)-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O(4)-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O(4)-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O(4)-nBudT and O(4)-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O(4)-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.

Research question Frequent and mild scrotal heat stress (fmSHS) often occurs in humans. The aim of this study was to determine the impact of fmSHS on natural fertility, the IVF–embryo transfer (IVF–ET) process and the offspring sex ratio. Design Male mice were randomly divided into four groups: no scrotal heat stress (SHS) (controls) and those subjected to SHS at 37°C, 39°C or 41°C for 30 min once a week for 5 consecutive weeks. The testis, epididymis and sperm quality were assessed to evaluate the effects of different degrees of SHS, to establish an fmSHS model. Then, natural fertility, IVF–embryo transfer (IVF–ET) results, embryo development, offspring sex ratio and the X/Y chromosome-bearing sperm ratio were examined. Results SHS at 39°C and 41°C caused mild impairment to spermatozoa, leading to a phenotype similar to oligoasthenozoospermia in humans. Given that most SHS conditions in humans are close to body temperature, SHS of 39°C was adopted to build the fmSHS model. fmSHS reduced the fertilization rate, impaired on-time development and reduced the implantation rate of the embryos in the IVF–ET process, but it did not affect the development or function of blastocysts. The fmSHS mice produced more Y chromosome-bearing spermatozoa and propagated more male offspring. Conclusions fmSHS not only reduced the fertilization ability of spermatozoa but also influenced their function beyond fertilization, in addition to changing the offspring sex ratio. These results may help to shed new light on the infertility treatment of males with scrotal heat risk and the health concerns of offspring propagated from these fathers.

Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O(6)-alkyl-2′-deoxyguanosine (O(6)-alkyl-dG) and minor-groove N(2)-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O(6)-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O(6)-alkyl-dG lesions. O(6)-alkyl-dG lesions mainly induced G→A mutations that were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O(6)-alkyl-dG lesions. Depletion of Pol ν alone reduced mutations only for O(6)-nBu-dG, and sole loss of Pol θ attenuated the mutation rates for O(6)-nBu-dG and O(6)-pyridyloxobutyl-dG. Replication across the two N(2)-alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.

Tumor-associated macrophages (TAMs), which display a tumor-supportive M2 phenotype, are closely related to tumor growth and metastasis. The reprogramming of TAMs toward a tumoricidal M1 profile has emerged as an attractive strategy for cancer immunotherapy. In this study, we found that the intratumoral injection of PcrV protein, a component of the Pseudomonas aeruginosa type 3 secretion system, suppressed tumor growth and increased apoptosis, inducible nitric oxide synthase (iNOS) expression, and the percentage of M1-polarized TAMs in tumor tissues. Furthermore, the intratumoral injection of PcrV-primed macrophages exerted a similar tumoricidal effect. In vitro analyses revealed that PcrV reeducated TAMs toward an antitumoral M1 phenotype and augmented their nitric oxide (NO)-mediated cytotoxicity against cancer cells. Mechanistically, we found that these effects were dependent on the activation of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-mediated regulation of a PI3K/AKT/mTOR-glycolysis-NO feedback loop via direct interaction with TLR4. Collectively, these results revealed a potential role for PcrV in cancer immunotherapy through the targeting of TAM plasticity.

RNA guanine quadruplexes (rG4) assume important roles in post-transcriptional regulations of gene expression, which are often modulated by rG4-binding proteins. Hence, understanding the biological functions of rG4s requires the identification and functional characterizations of rG4-recognition proteins. By employing a bioinformatic approach based on the analysis of overlap between peaks obtained from rG4-seq analysis and those detected in >230 eCLIP-seq datasets for RNA-binding proteins generated from the ENCODE project, we identified a large number of candidate rG4-binding proteins. We showed that one of these proteins, G3BP1, is able to bind directly to rG4 structures with high affinity and selectivity, where the binding entails its C-terminal RGG domain and is further enhanced by its RRM domain. Additionally, our seCLIP-Seq data revealed that pyridostatin, a small-molecule rG4 ligand, could displace G3BP1 from mRNA in cells, with the most pronounced effects being observed for the 3′-untranslated regions (3′-UTR) of mRNAs. Moreover, luciferase reporter assay results showed that G3BP1 positively regulates mRNA stability through its binding with rG4 structures. Together, we identified a number of candidate rG4-binding proteins and validated that G3BP1 can bind directly with rG4 structures and regulate the stabilities of mRNAs.

Emodin is widely present in Chinese herbs with broad application prospects, however, the conflicting reports of its hepatotoxicity have created a concern. It was therefore aimed to develop practical models to elucidate the outcome of CYP450 biotransformation on emodin. HepG2 and rat liver microsomes (RLM) coculture system was first utilized for prediction. It was found that emodin (35 μM)-mediated cytotoxicity was alleviated only when the cofactor of CYP450 NADPH (1 mM) was present. Similarly, both the pan-CYP450 inhibitor 1-aminobenzotriazole (ABT) (2 mM) and the heat-inactivated liver microsomes completely abolished the protective effect of RLM (0.75 mg/mL). Consistently, ABT significantly increased the toxicity of emodin in primary rat liver cells. Along similar lines, only the monohydroxylation metabolite M3 that accounted for neglectable amount of the whole metabolites showed similar toxicity to emodin, both M1 and M2 exhibited far less toxcity than emodin in THLE-2 cells. In vivo study further supported that ABT (50 mg/kg, s.c.) aggravated the hepatotoxicity of emodin (80 mg/kg, i.p.) on mice, as emodin treatment only mediated slight increase of liver index and histological score likely due to the metabolic detoxication of emodin, whereas ABT co-administration resulted in severe liver injury as reflected by the dramatic increase of the liver index value, serum ALT and AST levels, and histopathological score. Moreover, it was explored that ROS generation together with the electrophilicity of emodin contributed to its hepatotoxicity. These findings not only provided a clear evidence of the metabolic detoxification of emodin, but also shed a light on the hepatotoxic mechanisms of emodin, which would lay a solid foundation for the rational application of emodin in the future.

Fish of genus Erythroculter (Cypriniformes, Cyprinidae, Abramidinae) are important commercial freshwater species in China. In this work, morphological characters and gene sequences of mitochondrial CO I and Cyt b were analysed to investigate the germplasm differences among E. mongolicus, E. dabryi and E. ilishaeformis. Results demonstrated that about countable trait, there were six traits (scale in/ above/ below lateral line, gill‐raker in first left gill‐arch and branched fin‐ray in anal/ pectoral fin) showed significant differences. Gene fragments of CO I and Cyt b was 905 bps and 1,142 bps respectively. In CO I, there were 54.3% base content of A + T, 14 haplotypes and 84 nucleotide variable sites. As to Cyt b, there were 56.3% base content of A + T, 20 haplotypes and 142 nucleotide variable sites. In both CO I and Cyt b, the highest and lowest nucleotide diversity was detected in E. ilishaeformis and E. mongolicus respectively. According to morphological distance, genetic distance, genetic differentiation coefficient (Fst) and gene flow, E. dabryi was grouped together with E. ilishaeformis firstly, and then clustered with E. mongolicus. These results might be useful in the conservation and management of fishery resources of these three species.

Pseudomonas aeruginosa is a virulent bacterium that secretes a variety of virulence factors that aid in establishing infections in individuals. Allicin, derived from garlic, has been shown to inhibit virulence factor production and biofilm formation in P. aeruginosa. However, the mechanisms underlying the allicin-mediated regulation of P. aeruginosa virulence remain unclear. In this study, we investigated the possible mechanisms underlying allicin-mediated virulence regulation in P. aeruginosa. The results showed that allicin attenuates the production of P. aeruginosa virulence-associated factors, such as elastase, pyocyanin, pyoverdine, and rhamnolipids, by inhibiting the rhl and pqs quorum-sensing systems. Further analysis revealed that the rhl and pqs systems play different roles during the allicin-mediated regulation process. Taken together, these results support the potential use of allicin as a therapeutic agent in controlling P. aeruginosa infection and associated mechanisms.

Coxsackievirus B3 (CVB3) is the predominant pathogen of viral myocarditis. In our previous study, we found that CVB3 caused abnormal lipid accumulation in host cells. However, the underlying mechanisms by which CVB3 disrupts and exploits the host lipid metabolism are not well understood. Sterol regulatory element binding protein 1 (SREBP1) is the major transcriptional factor in lipogenic genes expression. In this study, we demonstrated that CVB3 infection and nonstructural 2A protein upregulated and activated SREBP1a at the transcriptional level. Deletion analysis of SREBP1a promoter revealed that two regions, –1821/–1490 and –312/+217, in this promoter were both required for its activation by 2A. These promoter regions possessed several binding motifs for transcription factor SP1. Next, we used SP1-specific small interfering RNAs (siRNAs) to confirm that SP1 might be the essential factor in SREBP1a upregulation by 2A. Furthermore, we showed that MEK/ERK pathway was involved in the activation of SREBP1a by 2A and that blocking this signaling pathway with the specific inhibitor U0126 attenuated SREBP1a activation and lipid accumulation by 2A. Finally, we showed that inhibition of SREBP1 with siRNAs attenuated lipid accumulation induced by CVB3 infection and reduced virus replication. Moreover, inhibition of the MEK/ERK pathway also led to reduction of SREBP1a activation, lipid accumulation, and virus replication during CVB3 infection. Taken together, these data demonstrate that CVB3 nonstructural 2A protein activates SREBP1a at the transcription level through a mechanism involving MEK/ERK signaling pathway and SP1 transcription factor, which promotes cellular lipid accumulation and benefits virus replication. IMPORTANCE Coxsackievirus B3 (CVB3) infection is the leading cause of viral myocarditis, but effective vaccines and antiviral therapies against CVB3 infection are still lacking. It is important to understand the precise interactions between host and virus for the rational design of effective therapies. During infection, CVB3 disrupts and exploits host lipid metabolism to promote excessive lipid accumulation, which benefits virus replication. SREBP1 is the master regulator of cellular lipid metabolism. Here, we report that one of the viral nonstructural proteins, 2A, upregulates and activates SREBP1a. Furthermore, we find that inhibition of SREBP1 decreases CVB3 virus replication. These results reveal the regulation of SREBP1a expression by 2A and the roles of SREBP1 in lipid accumulation and viral replication during CVB3 infection. Our findings provide a new insight into CVB3 host interactions and inform a potential novel therapeutic target for this important pathogen.

Objective: To establish UPLC fingerprint of Lophatherum gracile Brongn and provide reference for quality evaluation of Lophatherum gracile Brongn. Method: Agilent ZORBAX Eclipse Plus C18 (2.1mm × 100mm, 1.8 μm) chromatographic column, Mobile phase acetonitrile-0.1% phosphoric acid water, gradient elution, flow rate 0.1 mL·min-1, column temperature 30°C and injection volume 1μL. Results: There are 20 common peaks in the chromatographic fingerprint of 10 batches of Lophatherum gracile Brongn at 270 nm, and the similarity is between 0.890-0.974. Conclusion: The UPLC fingerprint method established in this study is efficient, fast, specific and characteristic, and the sample information is rich, which can provide reference for the quality evaluation of Lophatherum gracile Brongn.

Blumea balsamifera (L.) DC is an ethnic medicine with a significant anti-inflammatory effect. At present, there were many reports on the anti-inflammatory efficacy of B. balsamifera, but the mechanism of its is rarely reported. Therefore, the method of network pharmacology has been adopted in this paper to predict the molecular mechanism of the anti-inflammatory effect of B. balsamifera. The active chemical constituents of B. balsamifera were screened by reference to the literature, TCMSP and TCMID. A target data set of active chemical components was established by traditional chinese medicine target, TCMSP and BATMAN-TCM database. The target of the active ingredients were introduced into the HIT and TTD to establish a potential target data set of the B. balsamifera active ingredients. A OMIM was used to screen for inflammation-related genes and protein targets to establish an inflammatory target dataset. The complex network map of “active ingredient-target-disease” was constructed using Cytoscape3.6.1 software. A PPI analysis database was used to construct a protein interaction network of B. balsamifera component targets and inflammatory targets. GO functional and KEGG pathway enrichment analysis were performed by the biological information annotation database. As a result, 12 active chemical components in B. balsamifera were screened. The corresponding target of 724 active compounds were retrieved. There are 33 signaling pathways and 28 biological processes that were directly or indirectly related to the anti-inflammatory effects of B. balsamifera. Through enrichment analysis, the main signaling pathways of B. balsamifera include TNF signaling pathway, Hepatitis B, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, etc. Finally, Network pharmacology provides new ideas and methods for the study of the anti-inflammatory mechanism of B. balsamifera.

Zizania latifolia is the second-most cultivated aquatic vegetable in China, because of its nutritional and health-giving properties, and its cultivation is of considerable economic importance. Plant chitinases, as members of the glycoside hydrolase family, are considered primarily to be proteins involved in plant response to biotic and abiotic stresses, although they are also involved in many other physiological and morphological processes. In order to analyze the expression of chitinase genes under abiotic stress conditions in Z. latifolia and to better understand the functionality of these genes in this plant, we carried out a BLAST search of the Z. latifolia genome database and phylogenetic analysis based on the neighbor-joining method. Eleven chitinase genes were identified (ZlChi1-11) and classified into the chitinase classes I, II and IV. Seedlings were exposed to various abiotic stresses including salt, heat, cold, drought and the stress hormone abscisic acid (ABA) and expression patterns of all 11 chitinase genes in Z. latifolia were determined by quantitative reverse-transcription PCR. The results showed that most ZlChis were differentially expressed under abiotic stress conditions. The expression patterns indicated that most ZlChis responded to abiotic stresses within 6 h, although some ZlChis reached peak expression at 12 h or 18 h of abiotic stress treatment. Expression of ZlChi2 and ZlChi5 was upregulated under each of the stress treatments, while that of ZlChi10 or ZlChi11 was not detected under any conditions. This is the first report of expression analysis of all members of the glycoside hydrolase family 19 genes in Z. latifolia under abiotic stress, which provides basic information and insights into the putative roles of these genes in response to abiotic stress. The results of this study should serve as the basis for further functional characterization of these gene families in Zizania latifolia and related members of the Poaceae.

Taxol-based chemotherapy is widely used as the first-line treatment for non-small cell lung cancer (NSCLC), however, the subsequent development of taxol-resistance is a major concern and challenge, resulting in tumor relapse and poor prognosis. Given the complex nature of taxol-resistance, we further delved into its mechanisms and demonstrated that CYP1B1 was associated to taxol response in taxol-resistant A549/Taxol cells. Compared to its parent A549 counterpart, A549/Taxol presented much higher level of CYP1B1, which was paralleled by increased aryl hydrocarbon receptor (AhR) expressions likely due to the long term taxol exposure and thereby allowed a subsequent up-regulation of CYP1B1. Inhibition of CYP1B1 by TMS [(E)-2,3',4,5'-tetramethoxystilbene], the specific CYP1B1 inhibitor, remarkably enhanced the sensitivity of A549/Taxol to taxol. Moreover, pre-incubation of taxol with human recombinant CYP1B1 did not affect drug toxicity in A549 cells, precluding the possibility of drug resistance ascribed to CYP1B1 due to directly inactivating taxol. Indeed, CYP1B1 is responsible for bio-transforming estrogen (E2) into the carcinogenetic metabolite that would inhibit microtubule stabilization induced by taxol and thereby compromising treatment efficacy. Remarkably, our data revealed potent CYP1B1 inhibition efficacy of 4-hydroxyemodin (HEM) as reflected by both molecular docking simulations and EROD assay, which posed HEM the advantage of breaking the vicious circle between E2 and CYP1B1, not only favoring to overcome taxol-resistance, but also offering long term benefit via circumventing carcinogenesis and tumor progression induced by E2. In addition to CYP1B1 inhibition, HEM notably inhibited P-gp activity and expression, a common feature of drug resistance, as well as significantly inactivated AKT/ERK pathways that contributed to the cell proliferation, migration, and drug resistance. Thus, HEM may act in concert to overcome taxol-resistance through comprehensive targeting three considered arms of drug-resistance mechanisms. Moreover, HEM profoundly resisted E2-stimulated cell migration in both A549 and A549/Taxol cells, a primary reason for tumor patients' mortality, as well as inflicted selective injury to A549/Taxol cells rather than normal lung cells, supporting HEM to be a promising agent for overcoming taxol-resistance in A549 cells.

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change &gt, 2 or &lt, 0.5, p-value &lt, 0.05, q-value &lt, 0.01), which also have abundant expression (read counts &gt, 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.

BACKGROUND At present, the treatment of coxsackievirus-induced myocarditis remains difficult. Berberine (BBR), an isoquinoline alkaloid isolated from traditional medicine herbs, exhibits significant anti-viral efficacy against various viruses. However, the underlying mechanism by which BBR controls CVB3 infection has not yet been reported. The purpose of this study was to investigate the anti-viral efficacy of BBR against CVB3 infection and its mechanism. MATERIAL AND METHODS In our experiments, the protein levels of VP1 and MAPKs signal pathway were measured by Western blot. The mRNA level of VP1 was measured by RT-PCR. The virus titers were determined by TCID50 assay. RESULTS We found that BBR treatment significantly decreased CVB3 replication in HeLa cells. In addition, the BBR treatment reduced the phosphorylation levels of JNK and p38 MAPK upon CVB3 infection in both HeLa cells and primary rat myocardial cells. CONCLUSIONS Taken together, these results suggest that BBR inhibits CVB3 replication through the suppression of JNK and p38 MAPK activation, shedding new light on the investigation of therapeutic strategies against CVB3-induced viral myocarditis.

The goal of this study was to perform a systematic transcriptome-wide analysis of mRNA–miRNA interactions and to identify candidates involved in the interplay between miRNAs and mRNAs that regulate chicken muscle growth. We used our previously published mRNA (GSE72424) and miRNA (GSE62971) deep sequencing data from two-tailed samples [i.e., the highest (h) and lowest (l) body weights] of Recessive White Rock (WRR) and Xinghua (XH) chickens to conduct integrative analyses of the miRNA–mRNA interactions involved in chicken skeletal muscle growth. A total of 162, 15, 173, and 27 miRNA–mRNA pairs with negatively correlated expression patterns were identified in miRNA–mRNA networks constructed on the basis of the WRRh vs. XHh, WRRh vs. WRRl, WRRl vs. XHl, and XHh vs. XHl comparisons, respectively. Ingenuity Pathway Analysis revealed that gene networks identified for the WRRh vs. XHh contrast were associated with developmental disorders. Importantly, the WRRh vs. XHh contrast miRNA–mRNA network was enriched in IGF-1 signaling pathway genes, including FOXO3. A dual-luciferase reporter assay showed that FOXO3 was a target of miR-142-5p. Furthermore, miR-142-5p overexpression significantly decreased FOXO3 mRNA levels and promoted the expression of growth-related genes. These data demonstrated that miR-142-5p targets FOXO3 and promotes growth-related gene expression and regulates skeletal muscle growth in chicken. Comprehensive analysis facilitated the identification of miRNAs and target genes that might contribute to the regulation of skeletal muscle development. Our results provide new clues for understanding the molecular basis of chicken growth.

The stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR-4) axis plays an important role during stem cell recruitment. SDF-1 can also bind the more recently described CXCR-7 receptor, but effects of SDF-1/CXCR-7 signaling on stem cell migrating to ischemic brain injury area are little known. In the present study, we investigated the effect of CXCR-7 on bone marrow mesenchymal stem cell (BMSC) migration toward SDF-1α in the cerebral ischemia/reperfusion (I/R) rat hippocampus. We cultured BMSCs from rats and characterized them using flow cytometry, immunocytochemistry, western blotting, and immunofluorescence to detect SDF-1α, CXCR-4, and CXCR-7 expression in third passage BMSCs (P3-BMSCs). We also prepared the model of transient cerebral I/R by four-vessel occlusion (4-VO), and BMSCs were transplanted into I/R rat brain via lateral ventricle (LV) injection (20μl, 1×10(6)/ml). After that, we examined the effect of BMSCs migration in the cerebral I/R rat hippocampus through Transwell chamber assay. Our results show that SDF-1α, CXCR-4, and CXCR-7 were expressed in P3-BMSCs. Moreover, SDF-1α expression was increased in I/R hippocampus. At 48h after transplant, green fluorescent BrdU-BMSCs were observed in transplant groups, but no green fluorescent BrdU-BMSCs were seen in medium group. Among BMSCs transplant groups, the number of BrdU-BMSCs positive cell was the highest in BMSC group. Treatment with AMD3100 and/or CXCR-7 neutralizing antibody decreased the number of BMSC migration. Collectively, these findings indicate that CXCR-4 and -7 receptors were co-expressed in BMSCs and synergistically promoted BMSC migration. The effect of CXCR-7 was stronger than that of CXCR-4. Moreover, BMSCs that migrated to hippocampus promoted the autocrine and paracrine signaling of SDF-1α.

Coxsackievirus and adenovirus receptors (CARs) are the common cellular receptors which mediate coxsackievirus or adenovirus infection. Receptor trap therapy, which uses soluble viral receptors to block the attachment and internalization of virus, has been developed for the inhibition of virus infection. In this study, we have constructed a pPIC3.5 K/CAR-Fc expression plasmid for the economical and scale-up production of CAR-Fc fusion protein in Pichia pastoris . The coding sequence of the fusion protein was optimized according to the host codon usage bias. The amount of the CAR-Fc protein to total cell protein was up to 10% by 1% methanol induction for 96 h and the purity was up to 96% after protein purification. Next, the virus pull-down assay demonstrated the binding activity of the CAR-Fc to coxsackievirus. The analyses of MTT assay, immunofluorescence staining and quantitative real-time PCR after virus neutralization assay revealed that CAR-Fc could significantly block coxsackievirus B3 infection in vitro . In coxsackievirus B3 infected mouse models, CAR-Fc treatment reduced mortality, myocardial edema, viral loads and inflammation, suggesting the significant virus blocking effect in vivo . Our results indicated that the P. pastoris expression system could be used to produce large quantities of bioactive CAR-Fc for further clinical purpose.

Pathogenic bacteria could adjust gene expression to enable their survival in the distinct host environment. However, the mechanism by which bacteria adapt to the host environment is not well described. In this study, we demonstrated that nucleoside diphosphate kinase (Ndk) of Pseudomonas aeruginosa is critical for adjusting the bacterial virulence determinants during infection. Ndk expression was down-regulated in the pulmonary alveoli of a mouse model of acute pneumonia. Knockout of ndk up-regulated transcription factor ExsA-mediated T3S regulon expression and decreased exoproduct-related gene expression through the inhibition of the quorum sensing hierarchy. Moreover, in vitro and in vivo studies demonstrated that the ndk mutant exhibits enhanced cytotoxicity and host pathogenicity by increasing T3SS proteins. Taken together, our data reveal that ndk is a critical novel host-responsive gene required for coordinating P. aeruginosa virulence upon acute infection.

During infection, bacteria might generate adaptive responses to facilitate their survival and colonization in the host environment. The alarmone guanosine 5'-triphosphate-3'-diphosphate (ppGpp), the levels of which are regulated by the RelA and SpoT enzymes, plays a critical role in mediating bacterial adaptive responses and virulence. However, the mechanism by which ppGpp regulates virulence-associated traits in Pseudomonas aeruginosa is poorly understood. To investigate the regulatory role of ppGpp, the ppGpp-deficient strain ΔRS (relA and spoT gene double mutant) and the complemented strain ΔRS(++) (complemented with relA and spoT genes) were constructed. Herein, we reported that the ΔRS strain showed decreased cytotoxicity towards A549 human alveolar adenocarcinoma cell lines and led to reduced mortality, lung edema and inflammatory cell infiltration in a mouse model of acute pneumonia compared to wild-type PAO1 and the complemented strain ΔRS(++). Subsequent analyses demonstrated that the ΔRS strain displayed reduced T3SS expression, decreased levels of elastase activity, pyocyanin, pyoverdin and alginate, and inhibited swarming and biofilm formation compared to PAO1 and the complemented strain ΔRS(++). In addition, the results demonstrate that ppGpp-mediated regulation of T3SS, virulence factor production, and swarming occurs in a quinolone quorum-sensing system-dependent manner. Taken together, these results suggest that ppGpp is required for virulence regulation in P. aeruginosa, providing new clues for the development of interference strategies against bacterial infection.

Pig (Sus scrofa) is an important organism for both agricultural and medical purpose. This study aims to investigate the S. scrofa transcriptome by the use of Roche 454 pyrosequencing. We obtained a total of 558 743 and 528 260 reads for the back-leg muscle and ovary tissue each. The overall 1 087 003 reads give rise to 421 767 341 bp total residues averaging 388 bp per read. The de novo assemblies yielded 11 057 contigs and 60 270 singletons for the back-leg muscle, 12 204 contigs and 70 192 singletons for the ovary and 18 938 contigs and 102 361 singletons for combined tissues. The overall GC content of S. scrofa transcriptome is 42.3% for assembled contigs. Alternative splicing was found within 4394 contigs, giving rise to 1267 isogroups or genes. A total of 56 589 transcripts are involved in molecular function (40 916), biological process (38 563), cellular component (35 787) by further gene ontology analyses. Comparison analyses showed that 336 and 553 genes had significant higher expression in the back-leg muscle and ovary each. In addition, we obtained a total of 24 214 single-nucleotide polymorphisms and 11 928 simple sequence repeats. These results contribute to the understanding of the genetic makeup of S. scrofa transcriptome and provide useful information for functional genomic research in future.

Adipose triglyceride lipase (ATGL) is a crucial lipase that catalyzes triglyceride hydrolysis. In this study, reverse transcription (RT)-polymerase chain reaction, rapid amplification of cDNA ends, and mRNA real-time analysis were performed to characterize the ATGL gene in zebra finch and Java sparrow. Two cDNA of the zebra finch ATGL (zfATGL) gene were 1877 and 1626 bp were obtained; both encoded the same 481 amino acid protein. A 251 bp indel in the 3' UTR led to two variant transcripts. The zfATGL gene at chromosome 5 spanned over 28 Kb in length and comprised 9 exons. As in chicken, both the zfATGL gene and its intron 1 were much longer than mammals, which seemed to be one characteristic of ATGL gene in birds. The zfATGL gene was predominantly expressed in abdominal fat, subcutaneous fat, breast muscle, and leg muscle in both male and female. Two cDNA (1938 and 1885 bp) of the Java sparrow ATGL gene were also obtained, both of which encoded the same 481 amino acid protein. A 53 bp indel in 3' UTR led to two variant transcripts. The zfATGL gene was predominantly expressed in breast muscle, subcutaneous fat, abdominal fat, heart, and leg muscle. ATGL homology analysis showed that GASAG and GCGFLG motifs in the patatin region were conserved among 16 species. It was further indicated that the 251- and 53 bp indels at the 3' UTR of ATGL were potential binding sites for miRNA tgu-miR-2987. In conclusion, our study identified the full length cDNA and their variant transcripts of zfATGL and Java sparrow ATGL, and they were predominantly expressed in muscle, fat, and heart tissues.

The dopamine D2 receptor (DRD2) is a crucial mediator for normal physiological processes. We cloned the pig DRD2 gene, investigated its distribution in tissues and identified polymorphisms by RT-PCR, quantitative real-time PCR and direct sequencing. Two Yorkshire pigs from Guangdong Academy of Agricultural Sciences (Guangzhou, China) were selected to clone the gene and investigate its expression; 16 individuals from four pig breeds (Yorkshire, Landrace, small-ear spotted, and Xinchang) were used to scan the variations. The two transcripts (DRD2L and DRD2S), obtained through insertion or deletion of exon 5 and part of 3'UTR, were found to encode 444- and 415-amino acid proteins, respectively. The 574-bp indel in 3'UTR comprises five miRNA targeting sites, based on bioinformatics predictions. The pig DRD2 gene expresses predominantly in the pituitary gland, and then in oviducts and the hypothalamus. Both DRD2L and DRD2S mRNA were detected in cerebrum, cerebellum, hypothalamus, pituitary gland, back muscle, oviduct, uterus, and testis tissues; DRD2L was more abundant than DRD2S. The DRD2 gene is located on chromosome 9 and contains seven exons. Sixty-one different sequences were identified in this gene; among seven in the coding region, only one altered the encoded amino acid. These findings will help us understand the functions of the DRD2 gene in pigs.

Chicken growth performance provides direct economic benefits to the poultry industry. However, the underlying genetic mechanisms are unclear. The objective of this study was to identify candidate genes associated with chicken growth and investigate their potential mechanisms. We used RNA-Seq to study the breast muscle transcriptome in high and low tails of Recessive White Rock (WRRh, WRRl) and Xinghua chickens (XHh, XHl). A total of 60, 23, 153 and 359 differentially expressed genes were detected in WRRh vs. WRRl, XHh vs. XHl, WRRh vs. XHh and WRRl vs. XHl, respectively. GO, KEGG pathway and gene network analyses showed that CEBPB, FBXO32, FOXO3 and MYOD1 played key roles in growth. The functions of FBXO32 and FOXO3 were validated. FBXO32 was predominantly expressed in leg muscle, heart and breast muscle. After decreased FBXO32 expression, growth-related genes such as PDK4, IGF2R and IGF2BP3 were significantly down-regulated (P < 0.05). FBXO32 was significantly (P < 0.05) associated with carcass and meat quality traits, but not growth traits. FOXO3 was predominantly expressed in breast and leg muscle. In both of these tissues, the FOXO3 mRNA level in XH was significantly higher than that in WRR chickens with normal body weight (P < 0.05). In DF-1 cells, siRNA knockdown of FOXO3 significantly (P < 0.01) inhibited the MYOD expression and significantly up-regulated (P < 0.01 or P < 0.05) the expression of growth-related genes including CEBPB, FBXO32, GH, GHR, IGF1R, IGF2R, IGF2BP1, IGF2BP3, INSR, PDK1 and PDK4. Moreover, 18 SNPs were identified in FOXO3. G66716193A was significantly (P < 0.05) associated with growth traits. The sites C66716002T, C66716195T and A66716179G were significantly (P < 0.05) associated with growth or carcass traits. These results demonstrated that FOXO3 is a candidate gene influencing chicken growth. Our observations provide new clues to understand the molecular basis of chicken growth.

Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.

Natural resistance-associated macrophage protein 1 and 2 encoding genes (Nramp1 and Nramp2) are related to many diseases. We cloned the cDNA of chicken Nramp1 and Nramp2 genes, characterized their expression and polymorphisms, and investigated the association of some SNPs with resistance to salmonellosis. The Nramp1 cDNA was 1746 bp long and the Nramp2 cDNA was 1938 bp long. These cDNAs are similar to previously reported cDNAs, varying by two and one amino acids, respectively. The chicken Nramp1 gene expressed predominantly in liver, thymus and spleen in both females and males. The Nramp2 gene expressed in almost all tissues, but predominantly in breast muscle, leg muscle, cerebrum, cerebellum, lung, kidney, and heart in both females and males. We identified 45 SNPs and 2 indels in the chicken Nramp1 gene; three of 13 SNPs in the exons were missense mutations (Arg223Gln, Ala273Glu and Arg497Gln). Association analysis indicated that A24101991G is significantly associated with chicken salmonellosis resistance. These results will be useful for functional investigation of chicken Nramp1 and Nramp2 genes.

Prosaposin (PASP) is a sphingolipid hydrolysis protein that plays roles in both the nervous and reproduction systems. In this study, we cloned the pig PASP gene and studied its genomic organization, polymorphism, and expression pattern. Two PASP transcripts, TV1 (HQ245644) and TV2 (HQ245646), were identified in pig. TV1 was the complete transcript that encoded 527 amino acids, whereas TV2 was 9 bp shorter due to an exon 8 deletion. The pig PASP gene spanned over 34 kb in length on chromosome 14 (SSC14), and consisted of 15 exons and 14 introns. The pig PASP gene (TV1 and TV2) expressed predominantly in the cerebellum, lymphnode, pituitary, abdominal fat, hypothalamus, and cerebrum in both females and males. PASP TV1 expressed mainly in teh cerebrum, cerebellum, hypothalamus, pituitary, heart, subcutaneous fat, and foreleg muscle, while TV2 was expressed in the liver, spleen, lung, kidney, and lymphnode. In foreleg muscle, the predominant transcript was TV2 in males and TV1 in females. Some potential transcriptional elements were predicted in 5' flanking region (~3000 bp) of the PASP gene, and they were TATA boxes, RORE, Sp1, SRY, oct-1, Cdx A, and cap. Additionally, we identified 68 single-nucleotide polymorphisms and 9 indels in the pig PASP gene, and three single-nucleotide polymorphisms (C77932320T or L15F; C77928094T or P191L; A77917401G or K522R) were nonsynonymous substitutions. These results provide useful information for future functional investigations of the pig PASP gene.

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.